MASTER DUAL STAINING KIT

Principle Of The Analytical Method
The aim of the immunohistochemical staining is to convert into a visible stain the resulting antigen-antibody unit to be
studied in cells or tissues.
Thus, the Master Dual Staining Kit provides a set of highly specific and sensitive reagents which allow dual staining of
two different antigen-antibody units.
The kit is based on the use of a mix of two micropolymers, each labeled with different enzymes (Peroxidase and
Alkaline Phosphatase) each recognizing a specific immunoglobulin (antibody) developed in rabbit and mouse
respectively.
In case the reaction has occurred between the primary antibodies and antigens, the micropolymers specifically bind to
the resulted antigen-antibody unit. Due to enzyme label of the polymers, adding chromogen and its corresponding
substrates, precipitates of different colors are obtained which allows to microscopically detect the presence of a
specific antigens.
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Rev.: 2017-09-12
In the case of Master Dual Staining Kit, brown precipitates will be obtained for the antibodies developed in mouse
and red / purple color for rabbit antibodies.

Components and Reagents Included In The Kit:
• Peroxidase Blocking Reagent         MAD-021540Q-10        10mL
• POLYMER MIX                             MAD-001882QK-C        10mL

DAB:
DAB Substrate Buffer                     MAD-001812QK-A        15mL
DAB Chromogen Concentrate          MAD-001812QK-B        0,5mL

RED:
AP Substrate Buffer                       MAD-001818QK-A       2x15mL
AP Chromogen Concentrate            MAD-001818QK-B       0,5mL

1. Dewaxing and heat induced antigen retrieval2
a. Incubate slides with paraffin tissue sections in the oven at 60 ° C overnight.
b. Place the slides in the PT module using the flowing conditions:
– Pre-heat at 65ºC
– Dewax than followed by antigen retrieval 20 minutes a 95ºC

2. Detection and visualization (manual or automatic)
a. Wash in TBS – Tween 20 at RT
b. Apply 100 μl of Peroxidase Blocking Reagent on the tissue and incubate 10 min at RT
c. Wash 3 times in TBS.
d. Apply 100 μl of 1º Antibody* and incubate for 10 minutes** at RT
e. Wash 3 times in TBS.
f. Apply 100 μl of 2º Antibody* and incubate for 10 minutes** at RT
g. Wash 3 times in TBS.
h. Apply 100 μl of POLYMER MIX and incubate for 30 minutes at RT.
i. Wash 3 times in TBS.                                                                                                                                                               j. Mix a drop of DAB Chromogen Concentrate with 1 ml of DAB Substrate Buffer and applying the resulting
solution onto the tissue; incubate for 5 min at RT
k. Wash 3 times in distilled water
l. Mix a drop of AP Chromogen Concentrate with 2,5 ml of AP Substrate Buffer and applying the resulting
solution onto the tissue; incubate for 10 min at RT
m. Wash 3 times in distilled water